The overall goal of this work is to develop and validate a new suite of technologies that can rapidly and routinely generate circuit diagrams of nervous system tissue, sometimes called connectomes. The small size of neuronal processes and the synapses that connect them require that reconstruction be done at nanometer resolution;the distributed nature of neuronal connectivity requires reconstruction of large volumes, extending over a millimeter or more. Our method meets these two seemingly incompatible challenges by combining novel sectioning, electron microscopic imaging and reconstruction technologies. Together, these advances will allow us to acquire data and map circuits at least 1000-fold faster than has previously been possible. As a first test, we will reconstruct the retinal circuit of a mouse in its entirety. Enough is known about retinal structure and function to make this an appropriate tissue to validate the method. At the same time, this background will allow us to pose and solve important problems about neural circuits that will be directly applicable to the brain. We will then use the method to compare neural circuits in young adult and aged retina, providing insight into the structural basis of age-related neural decline. Finally, we will test the application of this connectomic method to human tissue. The new methods introduced here will transform neuroscience in several ways. First, it will allow elucidation of the structural underpinnings of brain function. It will also provide insight into how neural circuits are refined in early life and altered in old age. Second, applied to the ever increasing number of animal models of human behavioral disorders, it will help researchers delve into pathologies of cognition, behavior, and affect, some of which likely arise from miswiring of neural circuits. Finally, the method can be applied to any biological tissue where three-dimensional reconstruction of multiple large-volume specimens would be informative. PUBLIC HEALTH RELEVANCE: The connectome of each individual is probably unique, containing the traces of experiences and learned behaviors. Until we can acquire and decipher connectomic patterns, we will not know how acquired information is organized and encoded in the brain, or whether, as many suspect, miswiring underlies a variety of developmental, aging, and behavioral disorders. As the human genome project demonstrated, biomedical science can be transformed by scaling up and speeding up technologies to the point where they can provide comprehensive information about essential biology;our brain circuit reconstruction will be fast enough to compare entire circuits from multiple individuals at nanometer resolution.